Nucleic acids encoding cold sensitive mutant DNA polymerases

ABSTRACT

Provided are mutant DNA polymerase having at least one mutation which exhibit substantially reduced polymerase activity at 25° C. When compared to the same DNA polymerase without the at least one mutation and which exhibit normal or near-normal polymerase activity at optimum temperatures when compared to the same DNA polymerase without the at least one mutation. Also provided are amino acid sequences and nucleic acid sequences encoding such DNA polymerase, and vector plasmids and host cells suitable for the expression of these sequences. Also described herein are improved methods for performing polymerase chain reaction (PCR) amplification and other genetic manipulations and analyses using the mutant DNA polymerase of the invention.

This application is a divisional of U.S. patent application Ser. No.09/587,856, filed Jun. 6, 2000, now U.S. Pat. 6,214,557 incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is directed to thermostable DNA polymerase, andmore particularly, to novel mutants of Thermus aquaticus polymerases(Taq DNA polymerase). Specifically, the invention is directed to novelcold-sensitive mutants of Taq DNA polymerases and other thermostable DNApolymerases capable of catalyzing the amplification of polynucleotidesby PCR (the polymerase chain reaction) and exhibiting substantiallyreduced activity at temperatures in the range from room temperature (25°C.) to 42° C. When compared to the same polymerase without at least oneof the mutations, while retaining near-normal enzyme activity andfunctionality at the normal optimum temperature for the enzyme, 65 to72° C. The present invention is also directed to nucleic acid and aminoacid sequences encoding such mutants of Taq DNA polymerases, and vectorplasmids and host cells suitable for the expression of these DNAsequences. Also described herein is an improved method which providesfor an automatic hot start for performing polymerase chain reaction(PCR) amplification and other genetic analyses and manipulations usingthe DNA polymerases of the invention.

BACKGROUND OF THE INVENTION

PCR is a rapid and simple method for specifically amplifying a targetDNA sequence in an exponential manner. Saiki, et al. Science239:487-4391 (1988). Briefly, the method as now commonly practicedutilizes a pair of primers that have nucleotide sequences complementaryto the DNA which flanks the target sequence. The primers are mixed witha solution containing the target DNA (the template), a DNA polymeraseand dNTPS for all four deoxynucleotides (adenosine (A), tyrosine (T),cytosine (C) and guanine(G)). The mix is then heated to a temperaturesufficient to separate the two complementary strands of DNA. The mix isnext cooled to a temperature sufficient to allow the primers tospecifically anneal to sequences flanking the gene or sequence ofinterest. The temperature of the reaction mixture is then set to theoptimum for the thermophilic DNA polymerase to allow DNA synthesis(extension) to proceed. The temperature regimen is then repeated toconstitute each amplification cycle. Thus, PCR consists of multiplecycles of DNA melting, annealing and extension. Twenty replicationcycles can yield up to a million-fold amplification of the target DNAsequence. In some applications a single primer sequence functions toprime at both ends of the target, but this only works efficiently if theprimer is not too long in length. In some applications several pairs ofprimers are employed in a process commonly known as multiplex PCR.

The ability to amplify a target DNA molecule by PCR has applications invarious areas of technology e.g., environmental and food microbiology(Wernars et al., Appl. Env. Microbiol., 57:1914-1919 (1991); Hill andKeasler, Int. J. Food Microbiol., 12:67-75 (1991)), clinicalmicrobiology (Wages et al. J. Med. Virol., 33:58-63 (1991); Sacramentoet al., Mol. Cell Probes, 5:229-240 (1991)), oncology (Kumar andBarbacid, [Oncogene, 3:647-651 (1988); McCormick, Cancer Cells, 1:56-61(1989)), genetic disease prognosis (Handyside et al., Nature,344:768-770 (1990)), and blood banking and forensics (Jackson,Transfusion, 30:51-57 (1990)).

DNA polymerase obtained from the hot springs bacterium Thermus aguaticus(Taq DNA polymerase) has been instrumental in DNA amplification, DNAsequencing, and in related DNA primer extension techniques. The DNA andamino acid sequences described by Lawyer et al., J. Biol. Chem.,264:6427 (1989), GenBank Accession No. J04639, define the gene encodingThermus aquaticus DNA polymerase and the enzyme Thermus aquaticus DNApolymerase as those terms are used herein. The highly similar DNApolymerase (Tfl DNA polymerase) expressed by the closely relatedbacterium Thermus flavus is defined by the DNA and amino acid sequencesdescribed by Akhmetzjanov, A. A., and Vakhitov, V. A., Nucleic AcidsResearch 20: 5839 (1992), GenBank Accession No. X66105. These enzymesare representative of a family of DNA polymerases, also includingThermus thermophilus DNA polymerase, which are thermostable. Theseenzymes lack a 3′-exonuclease activity such as that which is effectivefor editing purposes in mesophilic DNA polymerases such as E. coli. DNApolymerase I, and phages T7, T3, and T4 DNA polymerases. ThermostableDNA polymerases which exhibit editing function are generally found inthermophilic archaebacteria such as Pyrococcus furiosus. Related DNApolymerases of this class are commonly known as Pfu, Pwo, Pfx, Vent, orDeep Vent.

The availability of thermostable DNA polymerases such as Taq DNApolymerase has both simplified and improved PCR. Taq DNA polymerase isstable up to 95° C. and its use in PCR has eliminated the necessity ofrepetitive addition of temperature sensitive polymerases after eachthermal cycle. Additionally, Taq DNA polymerase can extend DNA at highertemperatures which tends to prevent the non-specific annealing ofprimers and thus, has improved the specificity and sensitivity of PCR.

Although significant progress has been made in PCR technology, theamplification of non-target oligonucleotides due to side-reactions, suchas mispriming on non-target background DNA, RNA, and/or the primersthemselves, still presents a significant problem. This is especiallytrue in diagnostic applications where PCR is carried out in a milieucontaining complex background DNA while the target DNA may be present ina single copy (Chou et al., Nucleic Acid Res., 20:1717-1723 (1992)).

The temperature at which Taq DNA polymerase exhibits highest activity isin the range 62-72° C.; however, significant activity is exhibited atroom temperature, approximately 25° C. to 37° C. In a normal or “coldstart,” the primers may prime DNA extension at non-specific sequencesbecause the formation of only a few base pairs at the 3′-end of a primercan result in a stable priming complex. The result can be competitive orinhibitory products at the expense of the desired product. As an exampleof inhibitory product, structures consisting only of primer, sometimescalled “primer dimers” are formed by the action of DNA polymerase onprimers paired with each other, regardless of the true target template.The probability of undesirable primer-primer interactions increases withthe number of primer pairs in the reaction, as with multiplex PCR.During PCR cycling, these non-specific extension products can competewith the desired target DNA.

Further, it has been determined that side reactions often occur when allreactants are mixed at ambient temperature before thermal cycling isinitiated. One method for minimizing these side reactions is termed “hotstart” PCR. Many PCR analyses, particularly the most demanding ones,benefit from a hot start. About 50% of all PCR reactions show improvedyield and/or specificity if a hot start is employed, and in some cases ahot start is absolutely critical. These demanding PCR analyses includethose which have very low copy numbers of target (such as 1 HIV genomeper 10,000 cells), denatured DNA (many DNA extraction procedures includea boiling step, so that the template is single-stranded during reactionsetup), or contaminated DNA e.g., DNA from soil or feces and/or DNAcontaining large amounts of RNA. However, current methods of achieving ahot start are tedious, expensive, and/or have other shortcomings.

Hot start PCR may be accomplished by various physical, chemical, orbiochemical methods. In a physical hot start, the DNA polymerase or oneor more reaction components that are essential for DNA polymeraseactivity is not allowed to contact the sample DNA until all thecomponents required for the reaction are at a high temperature. Thetemperature must be high enough so that not even partial hybridizationof the primers can occur at any locations other than the desiredtemplate location, in spite of the entire genome of the cell beingavailable for non-specific partial hybridization of the primers. Thus,the temperature must be high enough so that base pairing of the primerscannot occur at template (or contaminating template) locations with lessthan perfect or near-perfect homology. This safe starting temperature istypically in the range of 50° C. to 75° C. and typically is about 10° C.hotter than the annealing temperature used in the PCR.

One physical way a hot start can be achieved is by using a wax barrier,such as the method disclosed in U.S. Pat. No. 5,599,660. See also Hebertet al., Mol. Cell Probes, 7:249-252 (1993); Horton et al.,Biotechniques, 16:42-43 (1994). Using such methods, the PCR reaction isset up in two layers separated by a 1 mm thick layer of paraffin waxwhich melts at about 56° C. There are several methods which may be usedto separate the reaction components into two solutions. For instance,all of the DNA is added, with 1×buffer but no dNTPs and no DNApolymerase enzyme, in a volume of 25 ml. One drop of melted wax is addedand the tubes are all heated to 60° C. for one minute to allow themelted wax to form a sealing layer after which the tubes are cooled sothe wax solidifies. Then a 25 ml mixture containing 1×buffer, all of thedNTPs, and the enzyme is added to each reaction. Finally, 1 drop of oilis added, to make 4 total layers. As the thermal cycler protocol heatsthe tubes to the first melting step (approximately 95° C.), the waxmelts and floats to mix with the oil layer, and the two aqueous layersmix by convection as the temperature cycles.

One common variation involving the use of a wax barrier is that thereaction components are assembled with no magnesium ions so that the DNApolymerase enzyme is inactive. The magnesium ion encased in a wax beadis then (or initially) added. A problem with these wax methods, however,is that the wax hardens after each PCR cycle. This makes sample recoveryextremely tedious, since the wax tends to plug the pipet tips used toremove the sample. This is true even if the samples are reheated to meltthe wax. Another potential problem is cross-contamination if tweezersare used to add wax beads, since slight contact between the tweezers andthe tube caps can move DNA template between samples before the PCRreactions start.

Another way to implement a hot start PCR is to use DNA polymerase whichis inactivated chemically but reversibly, such as AMPLITAQ GOLD™ DNApolymerase. This enzyme preparation, distributed by PE AppliedBiosystems, is distributed to users in inactivated form, but isreactivatable by heating. The required reactivation conditions, however,are extremely harsh to the template DNA: ten minutes at 95° C. and at anominal pH of 8.3 or lower results in reactivation of some 30% of theenzyme which is enough to start the PCR. See Moretti, et al.,BioTechniques 25: 716-722 (1998). Because this treatment depurinates DNAevery thousand bases or so, this enzyme can not be used to amplify DNAmore than a few kilobases in length. Accordingly, the use of this enzymeis most efficient when it is restricted to amplifying target DNA with alength of approximately 200 base pairs.

An additional way of implementing a hot start is to combine the Taq DNApolymerase enzyme with a Taq antibody before adding it to the reagent.This method employs a monoclonal, inactivating antibody raised againstTaq DNA polymerase. See Scalice et al., J. Immun. Methods, 172: 147-163(1994); Sharkey et al., Bio/Technology, 12:506-509 (1994); Kellogg etal., Biotechniques, 16: 1134-1137 (1994). The antibody inhibits thepolymerase activity at ambient temperature but is inactivated by heatdenaturation once the reaction is thermocycled, thus rendering thepolymerase active. Unfortunately, the antibodies currently available foruse in this method are not very efficient, and a 5 to 10-fold molarexcess must be used to effect the advantages of a hot start PCR. ForKlentaq-278, an amino-terminally deleted Thermus aquaticus DNApolymerase that starts with codon 279 which must be used at higherprotein levels for long PCR (up to ten times more protein than Taq DNApolymerase), the levels of antibody necessary for a hot start becomeextremely high and the denatured antibody protein retains someinhibition for longer PCR targets. The original developer of anti-Taqantibodies (Kodak, now Johnson & Johnson) uses a triple-monoclonalantibody mixture which is more effective but is not commerciallyavailable and has not been tested in long PCR.

These methods used for hot starts require inclusion of an oftenexpensive component (e.g., anti-Taq antibody) in the reaction mix andmay place some undesirable constraints on the performance of the PCRsuch as a relatively short time period between when a reagent isprepared and when it must be used, or a lower efficiency ofamplification. Therefore, it is usually preferable to perform physicalhot starts in PCR if at all feasible.

A low tech, inexpensive option is to add the enzyme, the magnesiumand/or the dNTPs to the reactions after they have heated up. Besidesbeing tedious and prone to error, this method commonly results incontamination and cross-contamination of PCR samples as the reactiontubes must be opened in the thermal cycler while they are hot.

Some workers believe they are doing a hot start when they set up PCRreactions in tubes on ice, then add the tubes to a thermal cycler blockpre-warmed to 95° C. Although some benefit arises from this method, theaddition of only a few nucleotides to a primer can take place everysecond during the fifteen seconds or more that the tubes warm from 0° C.to 25° C. This is enough to initiate unwanted competitive PCR forreactions that require a hot start. Also, if many tubes are involved inan experiment, the tubes placed in the block first are heated for alonger time period at 95° C. compared to the tubes placed later in theheating block thus resulting in a lack of reproducibility betweensamples.

Thermophilic DNA polymerases are commonly believed to have minimizedtheir mesotemperature activity during their evolution to optimizeactivity at around 70° C. According to this belief, it should not bepossible to further decrease their room temperature activity withoutseriously compromising either their high temperature activity or theirresistance to 95° C.

However, applicants conjectured the possibility that thermostable DNApolymerases could be mutated to a “cold-sensitive” phenotype in order todecrease polymerase activity at room temperature while not harming theactivity at the normal optimum extension temperature for PCR, nor thethermostability required for the melting step of each PCR cycle. Suchmutants are capable of catalyzing the PCR amplification and exhibitingsubstantially reduced activity at room temperature, yet near normalactivity at optimum reaction temperatures when compared to DNApolymerases without the mutations. Such mutant DNA polymerases would behighly useful in providing a hot-start capability and could be prepared,distributed and used without any additional steps or protocol changes.Thus, by adopting a cold sensitive DNA polymerase, end users could havethe advantages of a hot start for all of their PCR analyses, not justthe analyses that are first demonstrated to be problematic with a normalroom temperature start. Furthermore, “long and accurate” PCR (i.e.,employing longer target lengths and with enhanced fidelity) couldconveniently be provided the advantages of a hot start without tediousextra care or steps, and human STR typing and multiplex PCR will gain inreliability and efficiency. Such long and accurate PCR is described inBarnes, Proc. Natl. Acad. Sci. USA, 91:2216-2220 (1994) and in U.S. Pat.No. 5,436,149.

SUMMARY OF THE INVENTION

Among the several aspects of the invention, therefore, may be noted theprovision of cold-sensitive mutant DNA polymerases which exhibitsubstantially reduced activity at about 25° C. to 37° C. andsubstantially similar polymerase activity at 62-72° C. When compared toDNA polymerases without the mutations; the provision of such mutantswhich are useful for PCR amplification techniques from DNA templates andfrom single colonies of E. coli, single-stranded (linear) amplificationof DNA, cycle-sequencing, nucleic acid sequencing at elevatedtemperatures, DNA restriction digest filling, DNA labeling, in vivofootprinting and primer-directed mutagenesis. A further aspect of theinvention is the provision of recombinant amino acid and nucleic acidsequences, vectors and host cells which provide for the expression ofsuch mutant DNA polymerases. Yet another aspect of the invention is theprovision of an improved method of performing polymerase chainamplification catalyzed by the novel DNA polymerases. It is a furtheraspect of the present invention to provide methods using a coldsensitive DNA polymerase for PCR amplification from DNA templates andfrom single colonies of E. coli, single-stranded (linear) amplificationof DNA, nucleic acid sequencing, DNA restriction digest filling, DNAlabeling, in vivo footprinting and primer-directed mutagenesis.

Other aspects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, claims and accompanying drawings where:

FIG. 1 is a diagram of the plasmid pWB329, which carries the wild-typesequence of Klentaq-235 as the second gene of a 2-gene operon; the firstgene codes for NPTII, which kanamycin resistance. pWB329 was thetemplate for mutagenic PCR.

FIG. 2 is a diagram of the vector plasmid pWB302, which carries only theN-terminal portion of NPTII, and no DNA polymerase sequences at all.

FIG. 3 is a diagram of the library plasmids pWB302mk, which are theresult of cloning the PCR-mutagenized products as a library, into thesuitably digested vector plasmid pWB302.

FIG. 4 is a photograph of an agarose gel depicting the amplificationproducts obtained from a PCR amplification reaction using normal and twocold sensitive mutant DNA polymerases (Cs#1 and Cs#2) of the presentinvention. It should be noted that for this PCR reaction, normalpolymerase Klentaq-278 does not function well under cold start and warmstart conditions, but a manual hot start allows it to function.Conditions for conducting the PCR reaction are given herein.

FIG. 5 is a photograph of an agarose cell depicting the amplificationproducts obtained from a PCR amplification reaction using normal (lanes3 and 5) and a cold sensitive mutant DNA polymerase (Cs#3) (lanes 2 and4) of the present invention. Lane 1 contains a molecular weight standardladder. The PCR reactions in lanes 2 and 3 were performed by a manualhot start method and the PCR reactions in lanes 4 and 5 were conductedat room temperature (25° C.) and demonstrate the functional advantage ofthe mutant DNA polymerases of the present invention. Conditions forconducting the PCR reaction are given herein.

DETAILED DESCRIPTION

All publications, patents, patent applications or other references citedin this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor reference are specifically and individually indicated to beincorporated by reference.

Abbreviations and Definitions

The listed abbreviations and terms, as used herein, are defined asfollows:

bp is the abbreviation for base pairs.

Cs is the abbreviation for cold sensitive. As used herein, a “coldsensitive” enzyme is an enzyme displaying a phenotype in which theenzyme has reduced activity at temperatures below its optimum and normalor near-normal activity at the normal optimum temperature when comparedto the activity of its wild type enzyme at identical temperatures.

kb is the abbreviation for kilobase (1000 base pairs).

nt is the abbreviation for nucleotides.

ORF is the abbreviation for open reading frame.

Taq is the abbreviation for Thermus aquaticus.

Tfl is the abbreviation for Thermus flavus.

The amino acid residues are abbreviated herein according to their singleletters: A represents alanine; R represents arginine; N representsasparagine; D represents aspartic acid; C represents cysteine; Qrepresents glutamine; E represents glutamic acid; G represents glycine;H represents histidine; I represents isoleucine; L represents leucine; Krepresents lysine; M represents methionine; F represents phenylalanine;P represents proline; S represents serine; T represents threonine; wrepresents tryptophan; Y represents tyrosine; and V represents valine.

Klentaq-nnn is an amino-terminally deleted Thermus aquaticus DNApolymerase that starts with codon nnn+1, although the start codon andthe next codon may not match the wild type sequence because ofalterations to the DNA sequence to produce a convenient restrictionsite.

Klentaq-235 is a DNA polymerase having substantially the same amino acidsequence as Thermus aquaticus DNA polymerase, but excluding theN-terminal 235 amino acids, ±one residue as claimed in U.S. Pat. No.5,616,494, incorporated herein by reference.

Klentaq-278 is a DNA polymerase having substantially the same amino acidsequence as Thermus aquaticus DNA polymerase, but excluding theN-terminal 278 amino acids, as claimed in U.S. Pat. No. 5,436,149. Thecommon or commercial name for this DNA polymerase is Klentaql.

WT represents wild-type (full length) or the deletion of only threeamino acids, with no other known changes.

LA PCR is Long and Accurate PCR using an unbalanced mixture of two DNApolymerases, as claimed in U.S. Pat. No. 5,436,149.

ATCC is the abbreviation for American Type Culture Collection.

“Thermostable” is defined herein as having the ability to withstandtemperatures up to at least 95° C. for many minutes without becomingirreversibly denatured and the ability to polymerize DNA at optimumtemperatures of 55° C. to 75° C.

Processes of producing replicate copies of the same polynucleotide, suchas PCR or gene cloning, are collectively referred to herein as“amplification” or “replication.” For example, single or double strandedDNA may be replicated to form another DNA with the same sequence. RNAmay be replicated, for example, by a RNA directed RNA polymerase, or byreverse transcribing the RNA and then performing a PCR. In the lattercase, the amplified copy of the RNA is a DNA with the correlating orhomologous sequence.

The polymerase chain reaction (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using one or more primers,and a catalyst of polymerization, such as a DNA polymerase, andparticularly a thermally stable polymerase enzyme. Generally, PCRinvolves repeatedly performing a “cycle” of three steps: “melting”, inwhich the temperature is adjusted such that the DNA dissociates tosingle strands, “annealing”, in which the temperature is adjusted suchthat oligonucleotide primers are permitted match their complementarybase sequence using base pair recognition to form a duplex at one end ofthe span of polynucleotide to be amplified; and “extension” or“synthesis”, which may occur at the same temperature as annealing, or inwhich the temperature is adjusted to a slightly higher and more optimumtemperature, such that oligonucleotides that have formed a duplex areelongated with a DNA polymerase. The cycle is then repeated until thedesired amount of amplified polynucleotide is obtained. Methods for PCRamplification are taught in U.S. Pat. Nos. 4,683,195 and 4,683,202.

“Hot start PCR” is a PCR method that generally produces improvedreliability, improved products from low-copy targets, and/or cleaner PCRproducts. Template DNA and primers are mixed together and held at atemperature above the threshold of non-specific binding of primer totemplate. All of the PCR reaction components are added to the extensionreaction except one critical reagent which is withheld. The withheldreagent is usually the thermostable polymerase or the magnesium, but itcan also be, for instance, the triphosphates or the primers. Just priorto the cycling, the withheld reagent is added to allow the reaction totake place at higher temperature. Due to lack of non-specifichybridization of primers to template or to each other, the PCRamplification proceeds more efficiently as a result of the reduction orelimination of competing extensions at non-target locations. “Coldstart” and “room temperature start” are used interchangeably herein andwhen used to refer to PCR amplification, indicate that all the PCRreaction components needed for amplification are added to the templatenucleic acid sequence at 25° C.

When used to describe the temperature at which PCR amplification isconducted, “warm start” indicates that all the PCR reaction componentsneeded for amplification are added to the template nucleic acid sequenceat 30° C. or 37° C.

When referring to a particular protein such as a DNA polymerase, theterm “isolated” refers to the preparation of the protein which issubstantially free of contaminants.

When referring to a particular DNA polymerase, the term “polymeraseactivity” refers to the ability of the DNA polymerase to incorporatedNTPs or ddNTPS in a chain extension reaction. When referring to mutatedDNA polymerases, the term “substantially similar DNA polymeraseactivity” means the mutated polymerase exhibits at least 80% of thepolymerase activity of the same unmutated polymerase. When referring tomutated DNA polymerases, the term “substantially reduced DNA polymeraseactivity” means the mutated polymerase exhibits about 20% or less asmuch polymerase activity as the same unmutated polymerase.

“Reverse transcription” or “reverse transcribing” refers to the processby which RNA is converted into cDNA through the action of a nucleic acidpolymerase such as reverse transcriptase. Methods for reversetranscription are well known in the art and described for example inFredrick M. Ausubel et al. (1995), “Short Protocols in MolecularBiology,” John Wiley and Sons, and Michael A. Innis et al. (1990), “PCRProtocols,” Academic Press. “Stoffel fragment” refers to a DNApolymerase having substantially the same amino acid sequence as Therrnusaguaticus DNA polymerase but lacks the 5′ nuclease activity due to agenetic manipulation which results in the deletion of the N-terminal 289amino acids of the polymerase molecule. See Erlich et al., Science252:1643 (1991), incorporated herein by reference. “Thermus aquaticusDNA polymerase” or “Taq DNA polymerase” are used interchangeably torefer to heat stable DNA polymerases from the bacterium Thermusaquaticus and include all Taq mutants, natural and synthesized.

The procedures disclosed herein which involve the molecular manipulationof nucleic acids are known to those skilled in the art. See generallyFredrick M. Ausubel et al. (1995), “Short Protocols in MolecularBiology,” John Wiley and Sons, and Joseph Sambrook et al. (1989),“Molecular Cloning, A Laboratory Manual,” second ed., Cold Spring HarborLaboratory Press, which are both incorporated by reference.

Accordingly, the present invention is directed to novel mutant DNApolymerases which exhibit, when compared to the same unmutated DNApolymerases, substantially reduced polymerase activity at roomtemperature, but exhibit substantially similar polymerase activity atoptimum temperature. The mutated DNA polymerases exhibit about 20% orless as much polymerase activity at 25° C. and at least about 80% ormore as much polymerase activity at 68° C. when compared to the sameunmutated polymerases. In a preferred embodiment, the mutated DNApolymerase exhibit about 10% or less as much polymerase activity at 25°C. and at least about 80% or more as much polymerase activity at 68° C.when compared to the same unmutated polymerase; and even morepreferably, about 5% or less as much polymerase activity at 25° C. andat least 80% or more as much polymerase activity at 68° C. when comparedto the same unmutated polymerase. Most preferred are those mutated DNApolymerases which exhibit about 1.5% or less as much polymerase activityat 25° C. and at least about 80% or more as much polymerase activity at68° C. when compared to the same unmutated polymerase.

In one embodiment, the mutant DNA polymerases are thermally stable DNApolymerases. The thermostable enzyme may be obtained from varioussources and may be a native or recombinant protein. Some examples ofthermally stable DNA polymerases include, but are not limited to,Thermus aquaticus DNA polymerase, N-terminal deletions of Taq DNApolymerase such as Stof fel fragment DNA polymerase, Klentaq-235, andKlentaq-278; Thermus thermophilus DNA polymerase; Bacillus caldotenaxDNA polymerase; Thermus flavus DNA polymerase; Bacillusstearothermophilus DNA polymerase; and archaebacterial DNA polymerasessuch as Thermococcus litoralis DNA polymerase (also referred to asVent), Pfu, Pfx, Pwo, and Deep Vent. In one preferred embodiment, themutant DNA polymerases are Thermus aquaticus polymerases, morepreferably, full length or truncated Taq DNA polymerases, and even morepreferably, Klentaq-235 or Klentaq-278.

It will be appreciated that minor variations incorporated into the DNAencoding for, or the amino acid sequence as described herein, whichretain substantially the amino acid sequence as set forth in SEQ ID NO:2, and which do not significantly affect the thermostability of thepolymerase as included within the scope of the invention.

Those of ordinary skill in the art are aware that modifications in theamino acid sequence of a peptide, polypeptide, or protein can result inequivalent, or possibly improved, second generation peptides, etc., thatdisplay equivalent or superior functional characteristics when comparedto the original amino acid sequence. The present invention accordinglyencompasses such modified amino acid sequences. Alterations can includeamino acid insertions, deletions, substitutions, truncations, fusions,shuffling of subunit sequences, and the like, provided that the peptidesequences produced by such modifications have substantially the samefunctional properties as the naturally occurring counterpart sequencesdisclosed herein. Thus, for example, modified cell membrane-permeantpeptides should possess substantially the same transmembranetranslocation and internalization properties as the naturally occurringcounterpart sequence.

One factor that can be considered in making such changes is thehydropathic index of amino acids. The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein has been discussed by Kyte and Doolittle. See J. Mol. Biol.,157:105-132, (1982). It is accepted that the relative hydropathiccharacter of amino acids contributes to the secondary structure of theresultant protein. This, in turn, affects the interaction of the proteinwith molecules such as enzymes, substrates, receptors, DNA, antibodies,antigens, etc.

Based on its hydrophobicity and charge characteristics, each amino acidhas been assigned a hydropathic index as follows: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate/glutamine/aspartate/asparagine(−3.5); lysine (−3.9); and arginine (−4.5).

As is known in the art, certain amino acids in a peptide or protein canbe substituted for other amino acids having a similar hydropathic indexor score and produce a resultant peptide or protein having similarbiological activity, i.e., which still retains biological functionality.In making such changes, it is preferable that amino acids havinghydropathic indices within ±2 are substituted for one another. Morepreferred substitutions are those wherein the amino acids havehydropathic indices within ±1. Most preferred substitutions are thosewherein the amino acids have hydropathic indices within ±0.5.

Like amino acids can also be substituted on the basis of hydrophilicity.U.S. Pat. No. 4,554,101 discloses that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein. The following hydrophilicity values have been assigned to aminoacids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ±1); serine(+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5 ±1); alanine/histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); and tryptophan (−3.4). Thus, one amino acid in apeptide, polypeptide, or protein can be substituted by another aminoacid having a similar hydrophilicity score and still produce a resultantprotein having similar biological activity, i.e., still retainingcorrect biological function. In making such changes, amino acids havinghydropathic indices within ±2 are preferably substituted for oneanother, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

As outlined above, amino acid substitutions in the peptides of thepresent invention can be based on the relative similarity of the aminoacid side-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, etc. Exemplary substitutions that takevarious of the foregoing characteristics into consideration in order toproduce conservative amino acid changes resulting in silent changeswithin the present peptides, etc., can be selected from other members ofthe class to which the naturally occurring amino acid belongs. Aminoacids can be divided into the following four groups: (1) acidic aminoacids; (2) basic amino acids; (3) neutral polar amino acids; and (4)neutral non-polar amino acids. Representative amino acids within thesevarious groups include, but are not limited to: (1) acidic (negativelycharged) amino acids such as aspartic acid and glutamic acid; (2) basic(positively charged) amino acids such as arginine, histidine, andlysine; (3) neutral polar amino acids such as glycine, serine,threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and(4) neutral nonpolar amino acids such as alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, and methionine. It should benoted that changes which are not expected to be advantageous can also beuseful if these result in the production of functional sequences.

The present invention is further directed to amino acid sequences andnucleic acid sequences encoding such mutant DNA polymerases as well asvector plasmids and host cells suitable for the expression of these DNAsequences. Preferred host cells for the expression of these DNAsequences encoding the mutant DNA polymerases are bacterial cells,insect cells, yeast, plant cells and vertebrate animal cells.

A further aspect of the invention includes the DNA polymerases encodedby the polynucleotide sequences contained in the plasmids pWB329Cs#1 andpWB329Cs#2 deposited in accordance with the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure, on Aug. 23, 1999, at American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209,USA. These strains have the designations PTA-596 and PTA-597,respectively. As mentioned herein, for clarification, ATCC Deposit No.PTA-596 is identified in the ATCC Deposit as an E. coli K-12 bacterialstrain containing artificial plasmid pWB329Cs#1 whereas PTA-596 is morecorrectly described as artificial plasmid pWB302 ligated to Cs#1 asdescribed in Examples 1-4. Similarly, ATCC Deposit No. PTA-597 isidentified as an E. coli K-12 bacterial strain containing artificialplasmid pWB329Cs#2 whereas PTA-597 is more correctly described asartificial plasmid pWB302 ligated to Cs#2 as described in Examples 1-4.

Yet a further aspect of the invention is the DNA polymerase encoded bythe polynucleotide sequence contained in plasmid pWB302Cs#3. This DNApolymerase contains three amino acid changes as indicated in Table 3herein, at least one of which is critical to its cold-sensitivephenotype.

Uses for Mutant DNA Polymerases

The present invention provides mutant DNA polymerases which are usefulfor various PCR amplification techniques such as PCR amplification fromDNA templates and from single colonies of E. coli, single-stranded(linear) amplification of DNA, nucleic acid sequencing, DNA restrictiondigest filling, DNA labeling, mutation detection, and primer-directedmutagenesis.

The present invention is also directed to processes for amplifying aspecific nucleic acid sequence, preferably DNA or RNA, the processcomprising: (a) if the nucleic acid sequence is double stranded,separating the nucleotide strands and or melting all structures in thetemplate strands; (b) treating the single strands witholigodeoxyribonucleotide primers under conditions such that an extensionproduct of each primer is synthesized, using a mutated DNA polymerase ofthe present invention which extension product is complementary to eachDNA strand; and (c) separating the primer extension products from thetemplates on which they are synthesized to produce single strandedmolecules; and (d) repeating steps b and c at least once.

The mutant DNA polymerases of the present invention can also be used tosequence nucleic acid sequences of double and single stranded PCRtemplates. Such method of sequencing involves producing four mixtureseach consisting of nucleic acid sequence; a primer which will hybridizeto the nucleic acid sequence; one labeled dNTP and three unlabeleddNTPs; the mutant DNA polymerase; and a termination nucleotide. Each ofthe four mixtures contains a different termination ddNTP: ddATP, ddCTP,ddGTP and ddTTP. The sequence of the nucleic acid sequence can bedetermined by separating the amplification products of each mixture bygel electrophoresis and visualizing the labeled DNTP byaudioradiography. In one common alternative, the label is included onthe ddNTPs, each with a distinctive label, and all are included in onereaction and analyzed as one electophoresis sample, instead of fourseparate samples.

The present invention is further directed to processes of DNA labelingusing the novel mutant DNA polymerases to amplify the labeled DNAsequences. A nucleotide sequence can be labeled by providing a labelednucleotide comprising a reporter moiety, combining the labelednucleotide with the nucleic acid sequence template; amplifying thelabeled nucleic acid sequence by polymerase chain reaction using themutant DNA polymerases of the invention; and detecting the labelednucleic acid sequence. The labeled nucleotide can be contained in theDNA primer. Examples of various reporter moieties are radionucleotides,fluorophores or fluorochromes, peptides, antibodies, antigens, vitaminsand steroids.

The present invention is also directed to processes of in vivofootprinting using the mutant DNA polymerase to amplify the DNA. Ingeneral, analysis of the interaction of proteins with either DNA or RNAby in vivo footprinting involves first modifying the nucleic acids bythe footprinting reagent in situ. Footprinting reagents are chosen basedon how extensively the reactivity of a nucleic acid toward the modifyingagent is altered upon interaction with the binding protein of interest.The modifications are then visualized (i.e., the analysis of thereactivity of each nucleotide of the sequence of interest) usually byPCR. See Grange et al., Methods, (1997) 11:151-63. Accordingly, LM-PCRis utilized to visualize modifications in DNA molecules and RL-PCR isutilized to visualize modifications in RNA molecules. Both LM-PCR andRL-PCR involve ligating a linker to the unknown 5′-ends resulting fromthe in vivo footprinting analysis and exponentially amplifying theregion of interest. In LM-PCR, a blunt double-stranded end is createdusing a genespecific primer and a DNA polymerase. Then a partiallydoublestranded DNA linker with one blunt end is ligated to the bluntends using a DNA ligase. The strand onto which the linker has beenligated will then serve as a template for PCR amplification. Similarly,in RL-PCR, a single stranded RNA linker is ligated to the 5′ P-ends ofall RNA molecules using a RNA ligase. Then a cDNA copy of the sequenceof interest is synthesized using a reverse transcriptase which resultsin generating templates for PCR amplification. Lastly, amplifiedproducts from LM-PCR and RL-PCR are then labeled and sequenced foranalysis.

The present invention is also directed to processes of primer directedmutagenesis using the mutant DNA polymerases to amplify the mutatednucleic acid sequences having substitution mutations within the targetDNA sequence. The process of primer directed mutagenesis comprisescontacting a nucleic acid sequence with two mutated primers, where eachmutation is a mismatch when compared to the template sequence;amplifying using the novel mutated DNA polymerase; and allowing theamplified products to reanneal. The resulting nucleic acid moleculesamplified using these mismatched mutated primers have mismatched basesand have a double-stranded region containing a mutant strand. See Inniset al., “PCR Protocols”, Academic Press, 1990, pp 177-183.

The present invention is further directed to processes of DNArestriction digest filling using the novel mutant DNA polymerases toamplify the DNA. The mutant DNA polymerases are used in restrictiondigest filling to extend the 3′ ends resulting from digestion withrestriction enzymes for the purpose of producing 5′-sticky ends. Theprocess comprises separating the digested DNA strands; contacting each3′ end of the separated nucleic acid molecules witholigodeoxyribonuclotide primers; extending the 3′ ends using the novelmutated DNA polymerase to create blunt ends; and allowing the DNAstrands with the newly synthesized 3′ ends to reanneal to itscomplementary strand.

Construction of the Mutant Taq DNA Polymerases

The cold-sensitive DNA polymerases of the present invention are obtainedby systematically mutating a thermostable DNA polymerase. In general,the production of a mutated DNA polymerase typically involves thefollowing steps: obtaining and mutating a polynucleotide sequenceencoding the DNA polymerase; providing a DNA segment comprising themutated polynucleotides encoding a recombinant DNA polymerase; insertingthe DNA segment encoding the recombinant DNA polynucleotides into anexpression vector which is used to transform a host cell; and screeningto identify variants having the desired characteristics.

Mutation of the polynucleotide sequence can be accomplished by a varietyof methods well known in the art that are not critical to the invention.Mutation methods include, but are not limited to, chemical mutation,insertional mutation, deletion mutation, site directed mutation, randommutation, error prone PCR, oligonucleotide directed, and the like. Theresulting mutated gene encodes a recombinant, thermostable DNApolymerase.

In a preferred procedure, the polynucleotide sequence encodingthermostable Thermus aguaticus DNA polymerase, preferably Klentaq-235,is mutated by subjecting the polynucleotide sequence to randommutagenesis, preferably by an “error prone” PCR technique. Error pronePCR uses low-fidelity polymerization conditions to randomly introduce alow level of point mutations within a polynucleotide sequence and may beused to mutagenize a mixture of fragments of an unknown sequence. Seee.g., Leung et al., (1989) Technique, 1:11-15; Caldwell et al. (1992)PCR Methods Applic., 2:28-33; Gram et al., (1992) Proc. Natl. Acad.Sci., 89:3576-3580; Hawkins et al. (1992) J. Mol. Biol., 226:889-896.The mutated polynucleotide sequences are inserted into expressionvectors which are used to transform E. coli. The variants are thenscreened and those variants having the desired cold-sensitivecharacteristics are selected.

In general, preparation of an expression library consists of digestingthe nucleotide sequences encoding a mutated DNA polymerase and a vectorwith site specific restriction enzymes; and ligating the vector andmutated fragment together with the resulting insertion of the mutatedsequence adjacent to desired control and expression sequences. Theparticular vector employed will depend, in part, on the type of hostcell chosen for use in gene expression. Typically, a host-compatibleplasmid will be used containing genes for markers such as ampicillin ortetracycline resistance, and also containing suitable promoter andterminator sequences.

Specific nucleotide sequences in the vector are cleaved by site-specificrestriction enzymes such as NcoI and HindIII. Then, after optionalalkaline phosphatase treatment of the vector, the vector and targetfragment are ligated together with the resulting insertion of the targetcodons in place adjacent to desired control and expression sequences.

The DNA vector is then typically introduced into host cells, via aprocedure commonly known as transformation or transfection.Transformation of appropriate host cells may be performed using methodswell known in the art. The transformed host cells are cultured underfavorable conditions to effect the production of the recombinantthermostable DNA polymerase by expression of the gene and subsequentprotein production in the compatible transformed host. The controlsequences, expression vectors, and transformation methods are dependenton the type of host cell used to express the gene.

A host cell is transformed using a protocol designed specifically forthe particular host cell. For E. coli, a calcium treatment, Cohen, S.N., Proc. Natl. Acad. Sci. 69:2110 (1972), produces the transformation.Bacteria, e.g., various strains of E. coli, and yeast, e.g., Baker syeast, are frequently used as host cells for expression of DNApolymerase, although techniques for using more complex cells are known.See, e.g., procedures for using plant cells described by Depicker, A.,et al., J. Mol. Appl. Gen. (1982) 1:561.

Alternatively and more efficiently, electroporation of salt-free E. coliis performed after the method of Dower et al. (1988), Nucleic AcidsResearch 16:6127-6145. After transformation, the transformed hosts areselected from other bacteria based on characteristics acquired from theexpression vector, such as ampicillin resistance, and then thetransformed colonies of bacteria are further screened for the ability togive rise to high levels of isopropylthiogalactoside (IPTG)-inducedthermostable DNA polymerase activity. Colonies of transformed E. coliare then grown in large quantity and expression of the DNA polymerase isinduced for isolation and purification.

The expressed thermostable DNA polymerase is then isolated using avariety of methods known in the art and screened for desiredcharacteristics. Although a variety of purification techniques areknown, all involve the steps of disruption of the E. coli cells,inactivation and removal of native proteins and precipitation of nucleicacids. The DNA polymerase is separated by taking advantage of suchcharacteristics as its weight (centrifugation), size (dialysis,gel-filtration chromatography), or charge (ion-exchange chromatography).Generally, combinations of these techniques are employed together in thepurification process.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLES Example 1

Construction of pWB329

pWB250 containing a NPTII gene (from transposon Tn5) was digested withHindIII. After 90 minutes of digestion with HindIII, calf intestinealkaline phosphatase (2 units per 100 l) was added to the mixture for anadditional 30 minutes at 37° C. The plasmid pWB253 (see U.S. Pat. No.5,616,494) was digested with NcoI and one unit of Klenow fragment of DNApolymerase I in the presence of 50 mM of all 4 NTPs for 30 minutes at37° C. The pWB250 insert was then ligated into the vector pWB253 toproduce pWB329 containing a kanr gene (NPTIII) upstream of theKlentaq-235 gene in pWB253. pWB329 was used as the template DNA toextend the mutagenized sequences using non-mutagenic PCR in Example 2.

Example 2

Mutagenesis of Klentaq235

Error prone PCR was conducted using Klentaq-278 as the catalyzing DNApolymerase, no proof reader, and 0.5 mM Mn²⁺ in addition to 7.0 mM Mg²⁺ion in a reaction containing a PCR buffer of 250 μl of each dNTP, 50 mMTris pH 8.55, 16 mM ammonium sulfate. Template DNA consisted of thepolynucleotide sequence encoding Klentaq-235 DNA polymerase (plasmidpWB253; see U.S. Pat. No. 5,616,494) or genomic DNA from Thermusaquaticus. The PCR primers used in the mutagenesis reactions were KT85GCAGTACCGGGAGCTCACCAAGCTGAAGA (SEQ ID NO: 7) and Klentaq32 GCG AAG CTTACT ACT CCT TGG CGG AGA GCC AGT CC (SEQ ID NO: 8). Both KT85 andKlentaq32 span the C-terminal half Klentaq-235 (plasmid pWB253; see U.S.Pat. No. 5,616,494) thus concentrating the mutagenesis in the portion ofthe enzyme known to contain the catalytic functions for DNA polymerase.

The mutagenesis reactions were performed in triplicate, using threedifferent amounts of polymerase. For 15 cycles of mutagenic PCR(designated as m15), the template utilized was 10, 20 and 30 ng ofpWB253 in 100 μl volume. For 20 and 25 cycles of mutagenic PCR(designated as m20 and m25, respectively), the template utilized was 1,2 and 3 ng of genomic DNA from Thermus aquaticus in 100 μl volume.Mutagenic PCR cycling conditions were 60 seconds at 95° C. and 7 minutesat 65° C.

The products of the mutagenesis reactions m15, m20 and m25 were thenused as primers and extended utilizing a non-mutagenic high-fidelity PCRreaction using pWB329 as the template. The other primer utilized inthese reactions was oligonucleotide 4468 GGA TCT CGT CGT GAC CCA TGG CGATGC CTG CTT GCC (SEQ ID NO: 9) which spans the NcoI site in the NPTIIgene.

The triplicate PCR mutagenesis reactions (m15, m20 or m25) were pooledand precipitated with PEG. Primer oligonucleotides 4468 (SEQ ID NO: 9)and 1-2 ng plasmid pWB329 were added to each entire pellet in 200 μl ofPCR buffer containing 250 μM of each dNTP, 50 mM Tris pH 9.2, 16 mMammonium sulfate, 3.5 mM MgCl₂, 100 μg/ml BSA. Each reaction was dividedinto two tubes (tubes A and B) and 20 cycles of PCR were applied underthe following cycle parameters: 70 seconds at 96° C., 30 sec at 64° C.,and 7 minutes at 65° C. At cycle 12 of this final PCR, 24 pmoles ofprimer Klentaq32 (SEQ ID NO: 8) was added to tube B of each PCR reactionfor the pooled m15, m20 and m25. This addition enhanced the final yieldof the target 2 kb mutagenized PCR fragment. Each PCR reaction wasprecipitated twice with PEG in order to remove all dNTP and primer.

This resulted in the production of the entire DNA polymerase Klentaq-235together with a portion of the C-terminus of NPTII (kanamycinresistance). The NPTII portion enabled selection for PCR products duringthe cloning into expression vector pWB302 for the production ofexpression libraries of mutated polymerases in E. coli. Libraries wereprepared from each level of mutagenesis as described in Example 3 andscreened for DNA polymerase activity as described in Example 4.

Example 3

Preparation of Mutagenized Library pWB302mk

The pellets containing the mutated DNA sequences were resuspended inrestriction enzyme buffer NaTMS (50 mM NaCl, 10 mM Tris pH 7.9, 10 mMMgCl₂, 10 mM mercaptoethanol) and digested with NcoI and HindIII for 100minutes at 37° C. The expression vector pWB302 (FIG. 2) for cloning themutagenized PCR product was constructed as described in Barnes, W M,Gene 112:29-35 (1992). pWB302 is essentially a deletion of pWB305(Genbank Accession NO. M86847; Barnes 1992) between the SnaB1 and NcoIsites. pWB302 was digested with NcoI, HindIII and alkaline phosphatase.The digested target DNA and digested pWB302 were deprotonized by routinephenol extraction and ethanol precipitation and resuspended at aconcentration of approximately 0.2 μg/μl. The ligase reactions contained32.5 μl of water, 5 μl sticky-only T4 ligase buffer (40 μM rATP, 20 mMTris pH 7.9, 5 mM MgCl₂, 10 mM DTT), 10 μl target DNA, 2.5 μl vector and1 μl T4 DNA ligase. 10 μl was removed for the “before ligase” gelsample. The ligase reactions were incubated for 24 hours at 5° C. 10 μlwas removed for the “after ligase” gel sample. The remaining mixture wasprecipitated using ethanol. The “before ligase” and “after ligase”samples were run on an agarose gel to ensure proper ligation of themutagenesis products into pWB302.

The library of mutated DNA sequences inserted into the pWB302 vector isdesignated herein as pWB302mk. The region between primers KT85 (SEQ IDNO: 7) and Klentaq32 (SEQ ID NO: 8) contain the mutageneized sequences.In this system, the mutated polymerase genes are expressed at modestlevels as a second gene in an artificial operon of which kanR (NPTII) isthe first gene and a small portion of the kanR is included on theamplified DNA to be cloned. This provides for KanR selection so thatnone of the resulting library colonies is an empty vector.

Example 4

Identification of Cold Sensitive Mutants

Mutants with the desired phenotype were provisionally identified by asingle-colony assay. The single-colony DNA polymerase assay used byapplicants is a modification of the method of Sagner et al., Gene,97:119-123, 1991. Using a multipin replicator apparatus, E. colicolonies containing polymerase genes mutated by the methods describedabove were grown under standard conditions on nitrocellulose filters ata density of up to 384 per 8×12 cm filter (microtitre plate size).

Filters containing the colonies were overlaid over a minimal volume ofreaction buffer containing 50 mM Tris-HCl pH 7.9, 16 mM ammoniumsulfate, 2.5 mM magnesium chloride and 0.5% Triton X-100. The filterswere then heated at 68° C. for 15 minutes to inactivate endogenous E.coli DNA polymerases and any heat-sensitive mutants of Klentaq-235.

The filters were then underlaid with a minimal volume (2 ml) of reactionbuffer containing 20-40 mM all four dNTPs and a microcurie ofalpha-³²P-dATP, and incubated under either low temperature (37 or 42°C.) or high temperature (68° C.) conditions. To equalize the signal forcomparison between the two temperatures, the low temperature sampleswere incubated about 4-5 times longer than the high temperature samples.In an alternative procedure, low temperature screening was conducted at25° C. As will be apparent to those of ordinary skill in the art, whenscreening is conducted at 25° C., the increased incubation time neededto equalize the signals between high and low temperature conditions isincreased. After incorporation, the filters were washed with 5% TCA, 1%PPi and exposed to X-Ray film or phosphoimager until the wild-typesignals were easily detectable for each test temperature. Additionalequalization of the low-temperature-incubation signals versusnormally-high-temperature-incubation signals for unmutated controlcolonies was obtained by adjusting the exposure times of the final X-Rayfilm(s).

Colonies were selected on the basis of the difference in signalintensity generated between low and high temperature conditions. Moreparticularly, colonies were selected that produced no detectable signalunder low temperature conditions that produced full signal withwild-type, but produced a signal comparable to that generated bycontrol, unmutated colonies under high temperature conditions. Based onthese criteria, three colonies exhibiting the greatest cold sensitivitywere selected and designated Cs#1, Cs#2 and Cs#3.

E. coli bacteria strains containing plasmid pWB302 ligated to Cs#1 wasdesignated as pWB329Cs#1, and pWB302 ligated to Cs#2 was designated aspWB329Cs#2, and were deposited in accordance with the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure, on Aug. 23, 1999, at American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209,USA. These strains have the designation ATCC Deposit Nos. PTA-596 andPTA-597, respectively. For clarification, while ATCC Deposit No. PTA-596is identified in the ATCC Deposit as E. coli K-12 with artificialplasmid pWB329Cs#1, PTA-596 is more correctly described as artificialplasmid pWB302 ligated to Cs#1 as described in Examples 1-4. Similarly,while ATCC Deposit No. PTA-597 is identified in the ATCC deposit as E.coli K-12 with artificial plasmid pWB329Cs#2 whereas PTA-597 is morecorrectly described as artificial plasmid pWB2302 ligated to Cs#2 asdescribed in Examples 1-4.

The plasmid designated pWB329Cs#1, deposited with the ATCC as PTA-596(plasmid pWB302 ligated to Cs#1), contains a polynucleotide sequenceencoding a DNA polymerase having the mutations as indicated in Table 1.DNA sequence positions and amino acid sequence positions are numberedaccording to the DNA sequence and amino acid sequence of thefull-length, wild-type Taq DNA polymerase as shown as SEQ ID NO: 1 and2, respectively.

TABLE 1 Cold Sensitive Mutant DNA Polymerase #1 DNA Change Codon ChangeAmino Acid Change A 1912 T ATC to TTC I638F G 1974 T ATG to ATA M658I T2116 A TGG tc AGG W706R

The plasmid designated pWB329Cs#2, deposited with the ATCC as PTA-597(plasmid pWB302 ligated to Cs#2), contains a polynucleotide sequenceencoding a DNA polymerase having the mutations as indicated in Table 2.DNA sequence positions and amino acid sequence positions are numberedaccording to the DNA sequence and amino acid sequence of full-length,wild-type Taq DNA polymerase as shown as SEQ ID NO: 1 and 2,respectively.

TABLE 2 Cold Sensitive Mutant DNA Polymerase #2 DNA Change Codon ChangeAmino Acid Change T 2429 G GTG to GGG V810G A 2419 T ATG to TTG M807L A2042 G GAG to GGG E681G G 2124 T GAG to GAT E708D

pWB302Cs#3 (plasmid pWB302mk ligated to Cs#3) contains a polynucleotidesequence encoding a DNA polymerase having the mutations as indicated inTable 3. DNA sequence positions and amino acid sequence positions arenumbered according to the DNA sequence and amino acid sequence of afull-length, wild-type Taq DNA polymerase as shown as SEQ ID NO: 1 and2, respectively.

TABLE 3 Cold Sensitive Mutant DNA Polymerase #3 DNA Change Codon ChangeAmino Acid Change A 1842 T ATA to ATT I614I (no change) G 1876 A GAG toAAG E626K A 2069 G CAG to CGG Q690R A 2119 C ATT to CTT I707L

Using the purification procedures for Klentaq-235 and Klentaq-278 taughtin U.S. Pat. Nos. 5,616,494 and 5,436,149 (as incorporated herein byreference), colonies were expanded, induced with IPTG, the cells lysed,and the mutated DNA polymerases Cs#1, Cs#2 and Cs#3 were purified.

Example 5

PCR Amplification using Cs#1 and Cs#2

To determine if the cold-sensitive DNA polymerases of the presentinvention provide results under room temperature start conditionssimilar to those achieved using conventional thermal stable polymerasesand hot start conditions, Cs#1 and Cs#2 were compared to Klentaq-278using cold start, warm start, and manual hot start conditions. Thereactions were set up without magnesium in 50 ml volumes in 1×TAT buffercontaining 50 mM Tris-HCl pH 9.2, 16 mM ammonium sulfate, 0.1% Tween 20.The template consisted of 5 ng of human genomic DNA. One ml (about 0.7μg) of either Cs#1 polymerase, Cs#2 polymerase, or Klentaq-278 was usedper 50 ml of reaction mix. Each 50 ml reaction also contained 250 mM ofMg²⁺-free dNTPs. For the cold start and warm start conditions, magnesiumchloride (to a final level of 3.5 mM) was added before a 30 minutepre-incubation at the test temperature of 25, 30 or 37° C. To create amanual hot start, the reaction pre-incubation was at 30° C. withoutmagnesium and the magnesium chloride was added during the time period(approximately 5-7 minutes) that all the reactions were being incubatedat 68° C. before the first PCR cycle. Then, cycling conditions consistedof 45 cycles of 92° C. for 40 seconds, 67° C. for 30 seconds and 68° C.for 2 minutes.

Two target sequences of differing sizes were amplified. The humantyrosine hydroxylase gene (TH01) sequence consisting of 250 bp and thehuman chemokine receptor 5 (CCRS) sequence consisting of 513 bp wereamplified. The primer sets used are as follows:

THO1-1:

GTGGGCTGAAAAGCTCCCGATTAT (SEQ ID NO: 3)

THO1-2:

ATTCAAAGGGTATCTGGGCTCTGG (SEQ ID NO: 4)

CCR5-D5:

AGGTACCTGGCTGTCGTCCATGCTGTGTTT (SEQ ID NO: 5)

CCR5-D3:

GATGATGGGGTTGATGCAGCAGTGCGTCAT (SEQ ID NO: 6)

More particularly, for cold start PCR, 5 ml of 35 mM MgCl₂ were added to45 ml of reaction mix and pre-incubated at room temperature(approximately 25° C.) for 30 minutes. For warm start PCR, the procedurewas identical to that used for room temperature start except that the 30minute pre-incubation was carried out at 30° C. For hot start PCR, the45 ml reaction mixes were preincubated without Mg⁺⁺ at 30°0 C. for 30minutes. Then 2-4 minutes into the initial 5-7 minute warm-up (68° C.),5 ml of 35 mM MgCl₂ was added to each reaction mix. After cycling, theresulting amplification products were isolated using standard procedureswell known to those of ordinary skill in the art. See, e.g., Innis etal. (1990) “PCR Protocols, A Guide to Methods and Applications,”Academic Press, Inc, incorporated herein by reference.

Amplification products were visualized by agarose gel electrophoresisand ethidium bromide staining. Fifteen to eighteen ml of theamplification products were loaded into each well of the 1.4% agarosegel as shown in FIG. 4. The wells of the agarose gel containamplification products obtained under the conditions as shown in Table4. A 100 bp molecular weight ladder was loaded into well 1.Electrophoresis was carried out until the tracking dye had moved 5-7 cm.

TABLE 4 Lane PCR start conditions Enzyme Target DNA 2 cold start (25°C.) Cs#1 THO1 3 cold start (25° C.) Cs#2 THO1 4 cold start (25° C.)Klentaq-278 THO1 5 cold start (25° C.) Cs#1 CCR5 6 cold start (25° C.)Cs#2 CCR5 7 cold start (25° C.) Klentaq-278 CCR5 8 warm start (30° C.)Cs#1 THO1 9 warm start (30° C.) Cs#2 THO1 10 warm start (30° C.)Klentaq-278 THO1 11 warm start (30° C.) Cs#1 CCR5 12 warm start (30° C.)Cs#2 CCR5 13 warm start (30° C.) Klentaq-278 CCR5 14 hot start (68° C.)Cs#1 THO1 15 hot start (68° C.) Cs#2 THO1 16 hot start (68° C.)Klentaq-278 THO1 17 hot start (68° C.) Cs#1 CCR5 18 68° C. Cs#2 CCR5 1968° C. Klentaq-278 CCR5

As can be seen in FIG. 4, two of the cold-sensitive polymerases of thepresent invention, Cs#1 and Cs#2, produced sharp bands under either coldstart (Cs#1 in lanes 2 and 5; Cs#2 in lanes 3 and 6), warm start (Cs#1in lanes 8 and 11; Cs#2 in lanes 9 and 12), or hot start (Cs#1 in lanes14 and 17; Cs#2 in lanes 15 and 18) conditions. In contrast, the use ofstandard Klentaq-278 resulted in the formation of sharp bands only whenmanual hot start conditions are utilized (lane 16 and 19). For unknownreasons, the efficiency of amplification of the larger CCR5 targetsequence using the cold-sensitive polymerases under both roomtemperature and warm start conditions was lower than that observed forTHO1. Even at this lower efficiency, the results obtained using thecold-sensitive polymerases of the present invention are superior tothose obtained with Klentaq-278.

Example 6

PCR Amplification Using Cs#3

To determine whether the Cs#3 mutant polymerase of the present inventionprovides results under cold start conditions similar to those achievedusing conventional thermal stable polymerases and hot start conditions,Cs#3 polymerase was compared to Klentaq-278 using cold start and manualhot start conditions. The target human chemokine receptor 5 (CCR5)consisting of 513 bp was amplified. PCR was carried out using CCR5-D5(SEQ ID NO: 5) and CCR5-D3 (SEQ ID NO: 6) as primers for 35 cycles,using equal amounts (80 ng per 100 ul) of Klentaq-278 or Cs#3polymerase. All reactions contained 1.3 M betaine and Mg⁺⁺-free dNTPs.

To create a manual hot-start, PCR samples were pre-incubated at 30° C.for 30 minutes and prior to amplification, the reactions were warmed upat 68° C. for 10 minutes. Reactions 3 and 4 were pre-incubated at 30° C.without magnesium in 45 ml volumes of 1.11×TAT buffer (1×=50 mM Tris-HClpH 9.2, 16 mM ammonium sulfate, 0.1% Tween 20) at 30° C. for 30 minutes.Then 5 ml of 35 mM MgCl₂ was added to each reaction mix during the timeperiod that all the reactions were being incubated at 68° C. before thefirst PCR cycle, usually 2-4 minutes into the initial 5-7 minute warm-up(68° C.). For cold start PCR, 5 ml of 35 mM were added to the reactionmix and pre-incubated at room temperature (approximately 25° C.) for 30minutes. Amplification was conducted using the following cyclingconditions for both hot start and cold start: 45 cycles at 92° C. for 40seconds, 67° C. for 30 seconds and 68° C. for 2 minutes.

After cycling, the resulting amplification products were isolated usingstandard procedures well known to those of ordinary skill in the art.See, e.g., Innis et al. (1990) “PCR Protocols, A Guide to Methods andApplications,” Academic Press, Inc.

Amplified CCR5 products were visualized by agarose gel electrophoresisand ethidium bromide staining. Fifteen to eighteen ml of amplified CCR5products were loaded into wells 1-4 of an 1.4% agarose gel as shown inFIG. 5. The wells of the agarose gel contain amplification productsobtained under the conditions as shown in Table 5. A 100 bp molecularweight ladder was loaded into well 1. Electrophoresis was carried outuntil the tracking dye had moved 5-7 cm.

TABLE 5 Lane PCR Start Conditions Enzyme Target DNA 2 hot start (68° C.)Cs#3 CCRD5 3 cold start (25° C.) Cs#3 CCRD5 4 cold start (25° C.)Klentaq-278 CCRD5

As can be seen in FIG. 5, use of the Cs#3 polymerase produced sharpbands under either cold start (lane 4) or hot start (lane 2) conditions.In contrast, the use of standard Klentaq-278 resulted in the formationof sharp bands only when manual hot start (lane 3) conditions wereutilized. These results clearly demonstrate that the use of thecold-sensitive polymerases of the present invention eliminate the sidereactions observed with conventional thermal stable polymerases, thusproviding the advantages observed with hot start PCR without thepotential error and contamination problems currently associated with hotstart PCR.

In light of the detailed description of the invention and the examplespresented above, it can be appreciated that the several aspects of theinvention are achieved.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Further, the specific embodiments of the present inventionas set forth are not intended as being exhaustive or limiting of theinvention, and that many alternatives, modifications, and variationswill be apparent to those skilled in the art in light of the foregoingexamples and detailed description. Accordingly, this invention isintended to embrace all such alternatives, modifications, and variationsthat fall within the spirit and scope of the following claims. Whilesome of the examples and descriptions above include some conclusionsabout the way the invention may function, the inventors do not intend tobe bound by those conclusions and functions, but puts them forth only aspossible explanations.

9 1 2499 DNA Thermus aquaticus 1 atgaggggga tgctgcccct ctttgagcccaagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctgaagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagcctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggccccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggaggactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgcctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaaaaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttccgaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaaaagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtccgacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggagtgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggagaagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcaccgacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagggcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagccccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtgctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggccgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcgggggcttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggcgacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtggcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgagaggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttaccgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgcctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctcgaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctggaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggcaagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggagaagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccggacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggccacgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgcttgggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggactatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgggtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccccgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctctacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccaggccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagaccctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtgccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaacatgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttccccaggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgaggccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtgtatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgccaaggagtga 2499 2 810 PRT Thermus aquaticus 2 Met Arg Gly Met Leu Pro LeuPhe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His LeuAla Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg GlyGlu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys AlaLeu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys AlaPro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly ArgAla Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys GluLeu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly TyrGlu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu LysGlu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu TyrGln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp ArgLeu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp AspLeu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp LeuPro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg GluArg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu LeuHis Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu AlaPro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu SerArg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala AlaAla Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys AlaLeu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp LeuSer Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly AspAsp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr ThrPro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu ValAla 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu AlaGly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu ArgVal Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr GluLys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala LeuArg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg GluLeu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp LeuIle His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn GlnThr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn LeuGln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg ArgAla Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp GluAsn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys ValArg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg GlyTyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg MetAla Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met LysLeu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly AlaArg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala ProLys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu ValMet Glu Gly Val 805 810 3 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 3 gtgggctgaa aagctcccga ttat 24 4 24 DNAArtificial Sequence Description of Artificial Sequence Primer 4attcaaaggg tatctgggct ctgg 24 5 30 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 5 aggtacctgg ctgtcgtcca tgctgtgttt 30 6 30DNA Artificial Sequence Description of Artificial Sequence Primer 6gatgatgggg ttgatgcagc agtgcgtcat 30 7 29 DNA Artificial SequenceDescription of Artificial Sequence Primer 7 gcagtaccgg gagctcaccaagctgaaga 29 8 35 DNA Artificial Sequence Description of ArtificialSequence Primer 8 gcgaagctta ctactccttg gcggagagcc agtcc 35 9 36 DNAArtificial Sequence Description of Artificial Sequence Primer 9ggatctcgtc gtgacccatg gcgatgcctg cttgcc 36

We claim:
 1. A polynucleotide comprising a nucleotide sequence encodinga mutant DNA polymerase comprising at least one mutation, said mutantDNA polymerase exhibiting substantially reduced polymerase activity whencompared to the same polymerase without the at least one mutation at 25°C. and substantially similar polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C., saidpolynucleotide contained in the plasmid pWB302mk present in the hostcell selected from the group consisting of: ATCC Deposit Nos. PTA-596and PTA-597.
 2. The polynucleotide of claim 1 wherein the at least onemutation is selected from the group consisting of (1) an A to Tsubstitution at position 1912 of the nucleic acid sequence depicted inSEQ ID NO: 1; (2) a G to T substitution at position 1974 of the nucleicacid sequence depicted in SEQ ID NO: 1; and (3) a T to A substitution atposition 2116 of the nucleic acid sequence depicted in SEQ ID NO:
 1. 3.A DNA vector comprising the polynucleotide of claim
 2. 4. A host cellcomprising the vector of claim
 3. 5. The polynucleotide of claim 1wherein the at least one mutation is selected from the group consistingof (1) a T to G substitution at position 2429 of the nucleic acidsequence depicted in SEQ ID NO: 1; (2) an A to T substitution atposition 2419 of the nucleic acid sequence depicted in SEQ ID NO: 1; (3)an A to G substitution at position 2042 of the nucleic acid sequencedepicted in SEQ ID NO: 1; and (4) a G to T substitution at position 2124of the nucleic acid sequence depicted in SEQ ID NO:
 1. 6. A DNA vectorcomprising the polynucleotide of claim
 5. 7. A host cell comprising thevector of claim
 6. 8. A polynucleotide comprising a nucleotide sequenceencoding a mutant DNA polymerase comprising at least one mutation, saidmutant DNA polymerase exhibiting substantially reduced polymeraseactivity when compared to the same polymerase without the at least onemutation at 25° C. and substantially similar polymerase activity whencompared to the same polymerase without the at least one mutation at 68°C., wherein the at least one mutation is selected from the groupconsisting of (1) an A to T substitution at position 1912 of the nucleicacid sequence depicted in SEQ ID NO: 1; (2) a G to T substitution atposition 1974 of the nucleic acid sequence depicted in SEQ ID NO: 1; and(3) a T to A substitution at position 2116 of the nucleic acid sequencedepicted in SEQ ID NO:
 1. 9. A polynucleotide comprising a nucleotidesequence encoding a mutant DNA polymerase comprising at least onemutation, said mutant DNA polymerase exhibiting substantially reducedpolymerase activity when compared to the same polymerase without the atleast one mutation at 25° C. and substantially similar polymeraseactivity when compared to the same polymerase without the at least onemutation at 68° C., wherein the at least one mutation is selected fromthe group consisting of (1) a T to G substitution at position 2429 ofthe nucleic acid sequence depicted in SEQ ID NO: 1; (2) an A to Tsubstitution at position 2419 of the nucleic acid sequence depicted inSEQ ID NO: 1; (3) an A to G substitution at position 2042 of the nucleicacid sequence depicted in SEQ ID NO: 1; and (4) a G to T substitution atposition 2124 of the nucleic acid sequence depicted in SEQ ID NO:
 1. 10.A polynucleotide comprising a nucleotide sequence encoding a mutant DNApolymerase comprising at least one mutation, said mutant DNA polymeraseexhibiting substantially reduced polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. andsubstantially similar polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C., wherein the atleast one mutation is selected from the group consisting of (1) an A toT substitution at position 1842 of the nucleic acid sequence depicted inSEQ ID NO: 1; (2) a G to A substitution at position 1876 of the nucleicacid sequence depicted in SEQ ID NO: 1; (3) an A to G substitution atposition 2069 of the nucleic acid sequence depicted in SEQ ID NO: 1; and(4) an A to C substitution at position 2119 of the nucleic acid sequencedepicted in SEQ ID NO:
 1. 11. A DNA vector comprising the polynucleotideof claim
 10. 12. A host cell comprising the vector of claim
 11. 13. Arecombinant plasmid comprising plasmid pWB302mk containing a mutant DNApolymerase comprising at least one mutation, said mutant DNA polymeraseexhibiting substantially reduced polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. andsubstantially similar polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 14. Thepolynucleotide of claim 1 wherein said mutant DNA polymerase exhibitsabout 20% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 15. Thepolynucleotide of claim 14 wherein the mutant DNA polymerase exhibitsabout 10% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 16. Thepolynucleotide of claim 15 wherein the mutant DNA polymerase exhibitsabout 5% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 17. Thepolynucleotide of claim 16 wherein the mutant DNA polymerase exhibits atleast about 1.5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 18. Thepolynucleotide of claim 1 wherein the polymerase is a thermostablepolymerase.
 19. The polynucleotide of claim 18 wherein the thermostableDNA polymerase is a full length or truncated Taq polymerase.
 20. Thepolynucleotide of claim 18 wherein the thermostable DNA polymerase isselected from the group consisting of Thermus aquaticus DNA polymerase;N-terminal deletions of Taq DNA polymerase comprising a Stoffel fragmentDNA polymerase, Klentaq-235, and Klentaq-278; Thermus thermophilus DNApolymerase; Bacillus caldotenax DNA polymerase; Thermus flavus DNApolymerase; Bacillus stearothermophilus DNA polymerase; andarchaebacterial DNA polymerases comprising Thermococcus litoralis DNApolymerase, Pfu, Pfx, Pwo, and Deep Vent.
 21. The polynucleotide ofclaim 2 wherein said mutant DNA polymerase exhibits about 20% or less asmuch polymerase activity when compared to the same polymerase withoutthe at least one mutation at 25° C. and at least about 80% or more asmuch polymerase activity when compared to the same polymerase withoutthe at least one mutation at 68° C.
 22. The polynucleotide of claim 21wherein the mutant DNA polymerase exhibits about 10% or less as muchpolymerase activity when compared to the same polymerase without the atleast one mutation at 25° C. and at least about 80% or more as muchpolymerase activity when compared to the same polymerase without the atleast one mutation at 68° C.
 23. The polynucleotide of claim 22 whereinthe mutant DNA polymerase exhibits about 5% or less as much polymeraseactivity when compared to the same polymerase without the at least onemutation at 25° C. and at least about 80% or more as much polymeraseactivity when compared to the same polymerase without the at least onemutation at 68° C.
 24. The polynucleotide of claim 23 wherein the mutantDNA polymerase exhibits at least about 1.5% or less as much polymeraseactivity when compared to the same polymerase without the at least onemutation at 25° C. and at least about 80% or more as much polymeraseactivity when compared to the same polymerase without the at least onemutation at 68° C.
 25. The polynucleotide of claim 5 wherein said mutantDNA polymerase exhibits about 20% or less as much polymerase activitywhen compared to the same polymerase without the at least one mutationat 25° C. and at least about 80% or more as much polymerase activitywhen compared to the same polymerase without the at least one mutationat 68° C.
 26. The polynucleotide of claim 25 wherein the mutant DNApolymerase exhibits about 10% or less as much polymerase activity whencompared to the same polymerase without the at least one mutation at 25°C. and at least about 80% or more as much polymerase activity whencompared to the same polymerase without the at least one mutation at 68°C.
 27. The polynucleotide of claim 26 wherein the mutant DNA polymeraseexhibits about 5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 28. Thepolynucleotide of claim 27 wherein the mutant DNA polymerase exhibits atleast about 1.5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 29. Thepolynucleotide of claim 8 wherein said mutant DNA polymerase exhibitsabout 20% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 30. Thepolynucleotide of claim 29 wherein the mutant DNA polymerase exhibitsabout 10% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 31. Thepolynucleotide of claim 30 wherein the mutant DNA polymerase exhibitsabout 5% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 32. Thepolynucleotide of claim 31 wherein the mutant DNA polymerase exhibits atleast about 1.5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 33. Thepolynucleotide of claim 9 wherein said mutant DNA polymerase exhibitsabout 20% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 34. Thepolynucleotide of claim 33 wherein the mutant DNA polymerase exhibitsabout 10% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 35. Thepolynucleotide of claim 34 wherein the mutant DNA polymerase exhibitsabout 5% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 36. Thepolynucleotide of claim 35 wherein the mutant DNA polymerase exhibits atleast about 1.5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 37. Thepolynucleotide of claim 10 wherein said mutant DNA polymerase exhibitsabout 20% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 38. Thepolynucleotide of claim 37 wherein the mutant DNA polymerase exhibitsabout 10% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 39. Thepolynucleotide of claim 38 wherein the mutant DNA polymerase exhibitsabout 5% or less as much polymerase activity when compared to the samepolymerase without the at least one mutation at 25° C. and at leastabout 80% or more as much polymerase activity when compared to the samepolymerase without the at least one mutation at 68° C.
 40. Thepolynucleotide of claim 39 wherein the mutant DNA polymerase exhibits atleast about 1.5% or less as much polymerase activity when compared tothe same polymerase without the at least one mutation at 25° C. and atleast about 80% or more as much polymerase activity when compared to thesame polymerase without the at least one mutation at 68° C.
 41. A hostcell comprising the plasmid of claim 13.